REVIEW ARTICLE
Exosomal miRNAs as cancer biomarkers and therapeutic targets Arron Thind1* and Clive Wilson2 1 John Radcliffe Hospital, University of Oxford, Oxford, UK; 2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
Intercommunication between cancer cells and with their surrounding and distant environments is key to the survival, progression and metastasis of the tumour. Exosomes play a role in this communication process. MicroRNA (miRNA) expression is frequently dysregulated in tumour cells and can be reflected by distinct exosomal miRNA (ex-miRNA) profiles isolated from the bodily fluids of cancer patients. Here, the potential of ex-miRNA as a cancer biomarker and therapeutic target is critically analysed. Exosomes are a stable source of miRNA in bodily fluids but, despite a number of methods for exosome extraction and miRNA quantification, their suitability for diagnostics in a clinical setting is questionable. Furthermore, exosomally transferred miRNAs can alter the behaviour of recipient tumour and stromal cells to promote oncogenesis, highlighting a role in cell communication in cancer. However, our incomplete understanding of exosome biogenesis and miRNA loading mechanisms means that strategies to target exosomes or their transferred miRNAs are limited and not specific to tumour cells. Therefore, if ex-miRNA is to be employed in novel non-invasive diagnostic approaches and as a therapeutic target in cancer, two further advances are necessary: in methods to isolate and detect ex-miRNA, and a better understanding of their biogenesis and functions in tumour-cell communication. Keywords: microRNA; exosomes; exosome isolation; oncogenesis; tumour microenvironment; cell communication Responsible Editor: Edit Buza´s, Semmelweis University, Hungary.
*Correspondence to: Arron Thind, St Hugh’s College, St Margaret’s Road, Oxford, OX2 6LE, England, UK, Email:
[email protected] Received: 12 February 2016; Revised: 6 June 2016; Accepted: 9 June 2016; Published: 19 July 2016
ell communication is essential for tumorigenesis: individual tumour cells must interact with each other and host cells to survive, progress and metastasise. Through inter-tumour communication, a heterogeneous population of cells can co-operate and advance in a hostile environment. Local tumour-stromal communication facilitates manipulation of the microenvironment, optimising tumour growth, invasion and survival (1,2). Similarly, long-distance communication with stromal cells at distant host sites facilitates pre-metastatic niche formation, to promote colonisation and metastasis (3). However, the signals involved in tumour-stromal cell communication are yet to be fully elucidated. Deciphering these will facilitate the generation of novel, effective biomarkers and possibly provide a therapeutic benefit through targeting of these signals. It is becoming increasingly clear that tumour-derived exosomes (a form of endosome-derived extracellular vesicle [EV]) play an important role in this communication process through the transport of various proteins, lipids
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and nucleic acids in their membranous compartments (4). The discovery that exosomes can transfer microRNA (miRNA) between cells (5, Fig. 1) and the accompanying research developments in the miRNA field have sparked great interest in this area. miRNAs are short non-coding RNAs that target and repress complementary mRNAs, with roles in a number of cellular functions, such as differentiation, proliferation and cell cycle regulation (6). Due to the location of miRNA genes in chromosomal regions at cancer-associated genomic regions and fragile sites (unstable genomic regions that are often the sites of chromosomal rearrangements in cancer), expression is frequently dysregulated in tumour cells, leading to the upregulated expression of oncogenic miRNA and downregulation of tumour suppressor miRNA (7). The aberrant expression of cellular miRNA has been observed in cancer (7), and there is evidence that exosomal miRNA (ex-miRNA) expression is also altered (8). For this reason, could ex-miRNA be a potential cancer biomarker? Furthermore, could this dysregulation play a
Journal of Extracellular Vesicles 2016. # 2016 Arron Thind and Clive Wilson. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Journal of Extracellular Vesicles 2016, 5: 31292 - http://dx.doi.org/10.3402/jev.v5.31292
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Arron Thind and Clive Wilson
Nucleus Drosha Recipient cell Pol ll transcription
Pri-miRNA Cytoplasm
Pre-miRNA
Exportin 5 Exosomes DICER RAB27 MVB Pre-miRNA
RISC miRNA duplex
3-UTR
RISC
AAAAA
Translational Repression
ESCRT/ Ceramide Microvesicles
Mature miRNA
Early Endosome
RISC 3-UTR
AAAAA Translational Repression
Fig. 1. miRNA biogenesis and loading into exosomes. miRNA genes are transcribed by RNA polymerase II (Pol II), forming primiRNAs in the nucleus. The Drosha complex cleaves pri-RNA to pre-miRNA, which is exported to the cytoplasm via exportin 5. Further cleavage by the Dicer complex generates an intermediary miRNA duplex, of which one strand is incorporated into the RNA-induced silencing complex (RISC) to form mature miRNA, which targets complementary mRNA for translational repression. Inward budding of the early and late endosome forms exosomes. During this process, mature miRNA, some pre-miRNAs and other RNA molecules, proteins, and lipids are loaded into the exosomes. Within exosomes, loaded pre-miRNAs may be processed into mature miRNA. The exosome-loading process involves an endosomal-sorting complex required for transport (ESCRT) or ceramide-dependent mechanisms. The fusion of multivesicular bodies (MVBs) with the plasma membrane releases exosomes. This process is dependent on Rab GTPases (e.g. Rab27). The exosomal fusion with the plasma membrane of the recipient cell, or phagocytosis followed by membrane fusion, leads to the release of miRNA cargo into the cytosol and translational repression.
role in facilitating tumorigenesis? If so, ex-miRNA might provide a novel therapeutic target. Rapidly emerging methods in the field of exosome isolation and analysis have facilitated the identification of protein markers that distinguish exosomes from other EVs (9). The latter include microvesicles that bud off from the surface of the cell, including very large membranebound structures called oncosomes (10). In this review, there will be a specific focus on exosomes, since they are the best-characterised form of EV and most studies of miRNA-containing vesicles have targeted these structures. However, it is important to emphasise that often authors have not clearly distinguished between exosomes and other EVs, because of the current limitations with isolation procedures, although many methodologies employed typically separate away large oncosomes. Here, we will evaluate the technological and clinical feasibility of exmiRNA as a biomarker. Further, to determine its potential
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as a therapeutic target, the accuracy and relevance of experiments demonstrating the role of ex-miRNA in tumour progression will be critically discussed.
Ex-miRNAs as diagnostic markers Detection Current cancer biomarkers, such as prostate-specific antigen (PSA) and carbohydrate antigens, are plagued by problems such as false-negatives, false-positives and lack of tumour-type specificity (11). Consequently, tumour biopsy, a potentially invasive and damaging method, is the only definitive method of diagnosis. Non-exosomal, extracellular miRNA, bound to protein complexes (12), can be detected in biological fluids including blood, cerebrospinal fluid (CSF), breast milk, saliva and urine (13). Similarly, since their initial detection in secretions from mouse and human mast cell lines (5), ex-miRNAs have also been identified in bodily fluids (1417).
Citation: Journal of Extracellular Vesicles 2016, 5: 31292 - http://dx.doi.org/10.3402/jev.v5.31292
Exosomal miRNAs as cancer biomarkers
Further, exosomes have been found to provide a stable source of miRNA, preventing RNase degradation (18). In fact, exosomally derived miRNA has been demonstrated to remain stable at 208C for 5 years, is largely unaffected after 2 weeks at 48C and is resistant to freezethaw cycles (13). Therefore, exosomes are a source of miRNA that enables efficient storage and recovery in conditions that would normally degrade free miRNA. Due to its ease of access and stability, ex-miRNA has been proposed as a novel, minimally invasive tool for cancer diagnosis, with possible prognostic value. It inevitably suffers from some of the same problems as more conventional tumour markers. For example, it will almost certainly be secreted by other cell types, potentially masking cancer-specific signals. However, it is envisaged that, by profiling multiple ex-miRNA markers and by isolating exosomes using tumour-specific protein markers, it will be possible to improve on sensitivity and specificity, eliminating the issues faced with current cancer biomarkers. For effective biomarker analysis of exosomes, pure exosome samples are required. Currently, there are difficulties in the isolation of exosomes from other EVs found in bodily fluids. Exosomes are distinguished from other EVs, such as microvesicles, based on membrane composition, size and density. Whereas microvesicles are often 1001,000 nm
in diameter and originate from the plasma membrane, exosomes are around 40100 nm in diameter (19) and are enriched in proteins that are associated with the endocytic pathway, suggesting endosomal origin (20,21). Several methods have been employed to isolate exosomes (Fig. 2), the most common of which involves ultracentrifugation (UC) or ExoQuick precipitation. Both are fast and simple procedures, but relatively crude isolation methods for ex-miRNA measurements due to contaminating soluble proteins (particularly after ExoQuick isolation) and non-exosomal particles (22,23). Another method is immunoaffinity pulldown using anti-EpCAMcoupled microbeads, but not all exosomes express EpCAM (24). With this method, the failure to collect EpCAM-negative subtypes of exosomes and the presence of EpCAM-positive circulating tumour cells are likely to influence subsequent miRNA quantification. Few studies have adopted density gradient centrifugation (DGC), which allows the isolation of vesicles based on buoyant density. This approach has been shown to generate purer exosome samples than other methods (22,25, Fig. 2). However, the suitability of the density gradient method in a clinical setting is questionable, due to difficulties in upscaling and automating such a process.
Ultracentrifugation
ExoQuick
Immunoaffinity pulldown/MACS
OptiPrep density gradient
Dilute 5 ml plasma in 30 ml PBS and centrifuge to eliminate cell debris
Dilute 5 ml plasma in 30 ml PBS and centrifuge to eliminate cell debris
Incubation of 5 ml plasma with 50 ul anti-EpCAM conjugated magnetic microbeads (2 h; 4°C)
Dilute 5 ml plasma in 30 ml PBS and centrifuge to eliminate cell debris
Filtration (0.22 um)
Incubate with ExoQuick solution (12 h; 4°C)
Create 5–40% iodoxanol gradient by diluting a stock solution of OptiPrep. Layer sample on top
Ultracentrifugation (100,000–200,000; 3 h; 4°C)
Centrifuge (1,500 g; 5 min; 4°C) and discard the supernatant
Apply magnetic immune complexes onto a microcolumn placed in the magnetic field of a MACS separator. Discard unbound material
Discard supernatant and resuspend exosome pellet
Discard supernatant and resuspend exosome pellet
Dilute isolated exosomemicrobead complexes in lgG elution buffer and centrifuge (100,000 g; 1 h; 4°C)
Discard supernatant and resuspend exosome pellet Exosome purity: ** Exosome yield: *** Ease of use: ** Isolation time (h): 4 Hands on time (h):
